Biologic scaffolds composed of extracellular matrix (ECM) have been successfully used as templates for the constructive remodeling of numerous tissues in preclinical studies and human clinical applications. These ECM-based scaffolds have been surprisingly resistant to bacterial infection, even in clinical applications that are at high risk for bacterial contamination. The bacterial resistance property is associated with, and appears dependent upon, degradation of the ECM. In the absence of degradation, ECM scaffolds can actually support bacterial growth. The proposed work will investigate the mechanism by which ECM exerts antimicrobial activity, the tissue specificity of this activity, and the potential detrimental effect of a common method of processing biologic scaffolds, i.e., chemical crosslinking, upon the antimicrobial potential of the ECM. ECM scaffolds that are not processed by chemical crosslinking (e.g., carbodiimide) are rapidly degraded in vivo, and degradation of ECM scaffolds in vitro by chemical and physical methods has been shown to generate occult peptides that possess antimicrobial activity. The proposed studies will determine if the antimicrobial activity derived from degradation products of ECM scaffolds has site/tissue specificity. Stated differently, we will determine if the degradation products of dermal and liver ECM show their most potent activity against those bacteria that are commonly found in the skin and liver, respectively. Three specific aims are proposed: 1) To determine if antimicrobial degradation products of dermal and liver ECM scaffolds can be produced in vitro by methods that mimic physiologic conditions;2) To determine if degradation products of skin or liver ECM scaffolds have antimicrobial activity with site-specific activity;and 3) To isolate and purify antimicrobial peptides derived from dermal and liver ECM scaffolds. Clearly defined objectives and methods to successfully complete these specific aims are presented. The proposed work could potentially lead to the identification of novel antimicrobial peptides that could be produced in bulk for therapeutic use. This work will also elucidate the benefits of using non-chemically crosslinked ECM scaffolds for regenerative medicine applications, particularly in sites with high potential for bacterial infection. Finally, the proposed work will provide a better understanding of the role of ECM degradation in bacterial resistance associated with ECM scaffolds, as well as in mammalian innate immunity.